Methods and compositions for the treatment of sengers syndrome

ABSTRACT

The disclosure provides methods of preventing or treating Sengers syndrome in a mammalian subject, reducing risk factors associated with Sengers syndrome, and/or reducing the likelihood or severity of Sengers syndrome. The methods comprise administering to the subject an effective amount of an aromatic-cationic peptide to increase expression of AGK in subjects in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to U.S.Application No. 62/776,381, filed on Dec. 6, 2018, the contents of whichare incorporated herein in their entirety.

TECHNICAL FIELD

The present technology relates generally to compositions and methods forpreventing or treating Sengers syndrome, reducing risk factorsassociated with Sengers syndrome, and/or reducing the severity ofSengers syndrome. In particular, the present technology relates toadministering an effective amount of an aromatic-cationic peptide to asubject in need thereof to treat or prevent Sengers syndrome.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present technology.

Sengers syndrome, also known as hypertrophic cardiomyopathicmitochondrial DNA depletion syndrome-10 (MTDPS10), is a rare autosomalrecessive condition characterized by, but not limited to, cataracts,hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance,and lactic acidosis. The syndrome is caused by a homozygous or compoundheterozygous mutation in the mitochondrial lipid kinase acylglycerolkinase (AGK) gene.

Most children born with Sengers syndrome die within the first year oflife due to cardiac failure. However, a milder form of the disease alsoexists with some people surviving for multiple decades. Patients whosurvive the neonatal period and infancy manifest the chromic form withstable cardiomyopathy and myopathy and have normal mental developmentand intellect. Physical mobility is impaired due to muscular weakness inmost patients. Skeletal muscle biopsies of affected individuals showsevere mtDNA depletion. There is no known treatment for Sengerssyndrome.

SUMMARY

In one aspect, the present disclosure provides a method for treating orpreventing Sengers syndrome in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of thepeptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof. In some embodiments, the subject displays reduced levels ofacylglycerol kinase (AGK) expression compared to a normal controlsubject. In some embodiments, the peptide is administered daily for 6weeks or more. In some embodiments, the peptide is administered dailyfor 12 weeks or more. In some embodiments, the subject has beendiagnosed as having Sengers syndrome. In some embodiments, the Sengerssyndrome comprises one or more of cataracts, hypertrophiccardiomyopathy, reduced cardiac function as assessed by ejectionfraction, skeletal myopathy, exercise intolerance, lactic acidosis,neutropenia, tachydyspnea, nystagmus, eosinophilia, cervicalmeningocele, isolated Complex I deficiency, strabismus, hypotonia,hyporeflexia, delayed motor development, reduced AGK expression, reducedmitochondrial levels of glutamate carrier 1 (GC1), reduced mitochondriallevels of adenine nucleotide transporter type 1 (ANT1), reducedmitochondrial levels of adenine nucleotide transporter type 3 (ANT3),reduced mitochondrial levels of phosphate carrier (PiC), and reducedmitochondrial levels of translocase of the inner membrane 22 (TIM22)subunits (hTim22, Tim29, and hTim9). In some embodiments, the subject ishuman. In some embodiments, the peptide is administered orally,topically, systemically, intravenously, subcutaneously, intraocularly,intraperitoneally, or intramuscularly.

In some embodiments, the method further comprises separately,sequentially or simultaneously administering a cardiovascular agent tothe subject. In some embodiments, the cardiovascular agent is selectedfrom the group consisting of: a diuretic, an angiotensin-convertingenzyme (ACE) inhibitor, an angiotensin II receptor blocker or inhibitor,an angiotensin-receptor neprilysin inhibitor (ARNI), an I_(f) channelblocker or inhibitor, a beta blocker, an aldosterone antagonist, ahydralazine and isosorbide dinitrate, a diuretic, and digoxin.

In some embodiments, the pharmaceutically acceptable salt comprisesacetate, hydrochloride or trifluoroacetate salt.

In one aspect, the present disclosure provides a method for increasingthe expression of AGK in a mammalian subject in need thereof, the methodcomprising: administering to the subject a therapeutically effectiveamount of the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceuticallyacceptable salt thereof. In some embodiments, the expression of AGK inthe subject is about 2-5 fold less than the level of AGK expression in anormal control subject. In some embodiments, the peptide is administereddaily for 6 weeks or more. In some embodiments, the peptide isadministered daily for 12 weeks or more. In some embodiments, thesubject has been diagnosed as having, is suspected of having, or is atrisk of having Sengers syndrome. In some embodiments, the Sengerssyndrome comprises one or more of cataracts, hypertrophiccardiomyopathy, reduced cardiac function as assessed by ejectionfraction, skeletal myopathy, exercise intolerance, lactic acidosis,neutropenia, tachydyspnea, nystagmus, eosinophilia, cervicalmeningocele, isolated Complex I deficiency, strabismus, hypotonia,hyporeflexia, delayed motor development, reduced AGK expression, reducedmitochondrial levels of glutamate carrier 1 (GC1), reduced mitochondriallevels of adenine nucleotide transporter type 1 (ANT1), reducedmitochondrial levels of adenine nucleotide transporter type 3 (ANT3),reduced mitochondrial levels of phosphate carrier (PiC), and reducedmitochondrial levels of translocase of the inner membrane 22 (TIM22)subunits (hTim22, Tim29, and hTim9). In some embodiments, the subject ishuman. In some embodiments, the peptide is administered orally,topically, systemically, intravenously, subcutaneously, intraocularly,intraperitoneally, or intramuscularly

In some embodiments, the method further comprises separately,sequentially or simultaneously administering a cardiovascular agent tothe subject. In some embodiments, the cardiovascular agent is selectedfrom the group consisting of: a diuretic, an angiotensin-convertingenzyme (ACE) inhibitor, an angiotensin II receptor blocker or inhibitor,an angiotensin-receptor neprilysin inhibitor (ARNI), an I_(f) channelblocker or inhibitor, a beta blocker, an aldosterone antagonist, ahydralazine and isosorbide dinitrate, a diuretic, and digoxin.

In some embodiments, the pharmaceutically acceptable salt comprisesacetate, hydrochloride or trifluoroacetate salt.

In one aspect, the present disclosure provides, a method for reducingthe risk of Sengers syndrome in a mammalian subject having decreasedexpression of AGK compared to a normal control subject, the methodcomprising: administering to the subject a therapeutically effectiveamount of the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceuticallyacceptable salt thereof. In some embodiments, the pharmaceuticallyacceptable salt comprises acetate, hydrochloride or trifluoroacetatesalt.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing the Clinical Global Impression (CGI) severityof illness scores for a Sengers syndrome patient undergoing treatmentwith D-Arg-2′6′-Dmt-Lys-Phe-NH₂ according to the method described inExample 1. 1=Normal, not at all ill; 2=Borderline ill; 3=Mildly ill;4=Moderately ill; 5=Markedly ill; 6=Severely ill; 7=Among the mostextremely ill patients.

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the present technology are described below invarious levels of detail in order to provide a substantial understandingof the present technology. The definitions of certain terms as used inthis specification are provided below. Unless defined otherwise, alltechnical and scientific terms used herein generally have the samemeaning as commonly understood by one of ordinary skill in the art towhich this technology belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in an increase (e.g., normalization of)the expression of e.g., AGK in a subject in need thereof. In the contextof therapeutic or prophylactic applications, in some embodiments, theamount of a composition administered to the subject will depend on thetype and severity of the disease and on the characteristics of theindividual, such as general health, age, sex, body weight and toleranceto drugs. In some embodiments, it will also depend on the degree,severity and type of disease. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Thecompositions can also be administered in combination with one or moreadditional therapeutic compounds. In the methods described herein,aromatic-cationic peptides, such as D-Arg-2′6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate, hydrochlorideor trifluoroacetate salt, may be administered to a subject having one ormore signs, symptoms, or risk factors of Sengers syndrome, such as,e.g., cardiomyopathy, skeletal muscle abnormalities, neutropenia, slowdevelopment, weak muscle tone, increased levels of organic acids in theurine and blood, and/or frequent bacterial infections, such aspneumonia. For example, a “therapeutically effective amount” of thearomatic-cationic peptides includes levels at which a subject's levelsof AGK expression are increased after administration, and/or at whichthe presence, frequency, or severity of one or more signs, symptoms, orrisk factors of Sengers syndrome are reduced or eliminated. In someembodiments, a therapeutically effective amount reduces or amelioratesthe physiological effects of Sengers syndrome, and/or the risk factorsof Sengers syndrome, and/or the likelihood of developing Sengerssyndrome.

As used herein, the term “Sengers syndrome” refers to a genetic disordercaused by deficiencies in the mitochondrial lipid kinase acylglycerolkinase (AGK) gene. Signs and symptoms of Sengers syndrome include, butare not limited to, cataracts, hypertrophic cardiomyopathy, reducedcardiac function as assessed by ejection fraction, skeletal myopathy,exercise intolerance, lactic acidosis, neutropenia, tachydyspnea,nystagmus, eosinophilia, cervical meningocele, isolated Complex Ideficiency, strabismus, hypotonia, hyporeflexia, delayed motordevelopment, reduced AGK expression, reduced mitochondrial maximalrespiration, reduced mitochondrial levels of glutamate carrier 1 (GC1),reduced mitochondrial levels of adenine nucleotide transporter type 1(ANT1), reduced mitochondrial levels of adenine nucleotide transportertype 3 (ANT3), reduced mitochondrial levels of phosphate carrier (PiC),and reduced mitochondrial levels of translocase of the inner membrane 22(TIM22) subunits (e.g., hTim22, Tim29, and hTim9).

As used herein, the term “AGK” refers to the human acylglycerol kinaseencoded by the AGK gene, which is located on chromosome 7q34. AGK is asubunit of the mitochondrial translocase of the inner membrane 22(TIM22) protein import complex where it functions to regulate the importand assembly of mitochondrial carrier proteins. Mutations in the AGKgene cause Sengers syndrome.

As used herein, “isolated” or “purified” polypeptide or peptide refersto a polypeptide or peptide that is substantially free of cellularmaterial or other contaminating polypeptides from the cell or tissuesource from which the agent is derived, or substantially free fromchemical precursors or other chemicals when chemically synthesized. Forexample, an isolated aromatic-cationic peptide would be free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe agent. Such interfering materials may include enzymes, hormones andother proteinaceous and nonproteinaceous solutes.

As used herein, “normalizing” a subject's levels of AGK expressionrefers to altering the subject's levels of AGK expression in thedirection of “normal” or wild-type expression levels. For example,normalizing AGK expression levels in a subject with reduced AGKexpression compared to a normal subject refers to increasing the levelsof AGK expression. In some embodiments, normalizing AGK expression in asubject refers to attenuating or reducing the degree of reduced AGKexpression compared to e.g., an untreated control subject.

As used herein “increasing” a subject's AGK expression level meansincreasing the level of AGK in the subject (e.g., a subject's AGKexpression level such as RNA and/or protein level) in an organ ortissue. In some embodiments, increasing AGK expression level is anincrease by about 1%, about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or more. Alternatively, or additionally, in someembodiments, increasing AGK expression level is measured as anattenuation or reduction in the extent to which AGK expression isdecreased in a subject. In some embodiments, the AGK reduction isdecreased about 0.25 fold to about 0.5 fold, about 0.5 fold to about0.75 fold, about 0.75 fold to about 1.0 fold, or about 1.0 fold to about1.5 fold.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art.

As used herein, the term “simultaneous” therapeutic use refers to theadministration of at least two active ingredients by the same route andat the same time or at substantially the same time.

As used herein, the term “separate” therapeutic use refers to anadministration of at least two active ingredients at the same time or atsubstantially the same time by different routes.

As used herein, the term “sequential” therapeutic use refers toadministration of at least two active ingredients at different times,the administration route being identical or different. Moreparticularly, sequential use refers to the whole administration of oneof the active ingredients before administration of the other or otherscommences. It is thus possible to administer one of the activeingredients over several minutes, hours, or days before administeringthe other active ingredient or ingredients. There is no simultaneoustreatment in this case.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to therapeutic treatment, wherein the object is to prevent,reduce, alleviate or slow down (lessen) the targeted pathologiccondition or disorder. A subject is successfully “treated” for Sengerssyndrome if, after receiving a therapeutic amount of thearomatic-cationic peptides, such as D-Arg-2′6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate, hydrochlorideor trifluoroacetate salt, according to the methods described herein, thesubject shows observable and/or measurable reduction in or absence ofone or more signs and symptoms of Sengers syndrome, such as, e.g.,cardiomyopathy, skeletal muscle abnormalities, neutropenia, slowdevelopment, weak muscle tone, increased levels of organic acids in theurine and blood, and/or frequent bacterial infections, such aspneumonia. It is also to be appreciated that the various modes oftreatment or prevention of medical conditions as described are intendedto mean “substantial,” which includes total but also less than totaltreatment or prevention, and wherein some biologically or medicallyrelevant result is achieved. Treating Sengers syndrome, as used herein,also refers to treating reduced AGK expression levels characteristic ofthe syndrome, thereby causing an increase in AGK expression compared tothe subject's level of AGK expression prior to treatment.

As used herein, “prevention” or “preventing” of a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of symptoms of a disorder or condition in the treated samplerelative to an untreated control sample, or delays the onset or reducesthe severity of one or more symptoms of the disorder or conditionrelative to the untreated control sample. As used herein, preventingSengers syndrome includes preventing or delaying the initiation of,preventing, delaying, or slowing the progression or advancement of,and/or reversing the progression of Sengers syndrome. As used herein,prevention of Sengers syndrome also includes preventing a recurrence ofone or more signs or symptoms of Sengers syndrome.

Aromatic-Cationic Peptides

The present technology relates to methods and compositions forpreventing or treating Sengers syndrome in a subject in need thereof. Insome embodiments, the methods and compositions prevent one or more signsor symptoms of Sengers syndrome in a subject. In some embodiments, themethods and compositions increase the level of AGK expression in asubject. In some embodiments, the methods and compositions reduce thelikelihood that a subject with risk factors for Sengers syndrome willdevelop one or more signs or symptoms of Sengers syndrome.

The aromatic-cationic peptides are water-soluble and highly polar.Despite these properties, the peptides can readily penetrate cellmembranes. The aromatic-cationic peptides typically include a minimum ofthree amino acids or a minimum of four amino acids, covalently joined bypeptide bonds. The maximum number of amino acids present in thearomatic-cationic peptides is about twenty amino acids covalently joinedby peptide bonds. Suitably, the maximum number of amino acids is abouttwelve, more preferably about nine, and most preferably about six.

The amino acids of the aromatic-cationic peptides can be any amino acid.As used herein, the term “amino acid” is used to refer to any organicmolecule that contains at least one amino group and at least onecarboxyl group. Typically, at least one amino group is at the α positionrelative to a carboxyl group. The amino acids may be naturallyoccurring. Naturally occurring amino acids include, for example, thetwenty most common levorotatory (L) amino acids normally found inmammalian proteins, i.e., alanine (Ala), arginine (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),and valine (Val). Other naturally occurring amino acids include, forexample, amino acids that are synthesized in metabolic processes notassociated with protein synthesis. For example, the amino acidsornithine and citrulline are synthesized in mammalian metabolism duringthe production of urea. Another example of a naturally occurring aminoacid includes hydroxyproline (Hyp).

The peptides optionally contain one or more non-naturally occurringamino acids. Optimally, the peptide has no amino acids that arenaturally occurring. The non-naturally occurring amino acids may belevorotary (L-), dextrorotatory (D-), or mixtures thereof. Non-naturallyoccurring amino acids are those amino acids that typically are notsynthesized in normal metabolic processes in living organisms, and donot naturally occur in proteins. In addition, the non-naturallyoccurring amino acids suitably are also not recognized by commonproteases. The non-naturally occurring amino acid can be present at anyposition in the peptide. For example, the non-naturally occurring aminoacid can be at the N-terminus, the C-terminus, or at any positionbetween the N-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include α-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide is thederivatization of a carboxyl group of an aspartic acid or a glutamicacid residue of the peptide. One example of derivatization is amidationwith ammonia or with a primary or secondary amine, e.g. methylamine,ethylamine, dimethylamine or diethylamine. Another example ofderivatization includes esterification with, for example, methyl orethyl alcohol. Another such modification includes derivatization of anamino group of a lysine, arginine, or histidine residue. For example,such amino groups can be acylated. Some suitable acyl groups include,for example, a benzoyl group or an alkanoyl group comprising any of theC₁-C₄ alkyl groups mentioned above, such as an acetyl or propionylgroup.

The non-naturally occurring amino acids are suitably resistant orinsensitive to common proteases. Examples of non-naturally occurringamino acids that are resistant or insensitive to proteases include thedextrorotatory (D-) form of any of the above-mentioned naturallyoccurring L-amino acids, as well as L- and/or D-non-naturally occurringamino acids. The D-amino acids do not normally occur in proteins,although they are found in certain peptide antibiotics that aresynthesized by means other than the normal ribosomal protein syntheticmachinery of the cell. As used herein, the D-amino acids are consideredto be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides should have lessthan five, preferably less than four, more preferably less than three,and most preferably, less than two contiguous L-amino acids recognizedby common proteases, irrespective of whether the amino acids arenaturally or non-naturally occurring. Optimally, the peptide has onlyD-amino acids, and no L-amino acids. If the peptide contains proteasesensitive sequences of amino acids, at least one of the amino acids ispreferably a non-naturally-occurring D-amino acid, thereby conferringprotease resistance. An example of a protease sensitive sequenceincludes two or more contiguous basic amino acids that are readilycleaved by common proteases, such as endopeptidases and trypsin.Examples of basic amino acids include arginine, lysine and histidine.

The aromatic-cationic peptides should have a minimum number of netpositive charges at physiological pH in comparison to the total numberof amino acid residues in the peptide. The minimum number of netpositive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r). The minimum number of net positive chargesdiscussed below are all at physiological pH. The term “physiological pH”as used herein refers to the normal pH in the cells of the tissues andorgans of the mammalian body. For instance, the physiological pH of ahuman is normally approximately 7.4, but normal physiological pH inmammals may be any pH from about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively chargedamino acid (i.e., Glu) and four positively charged amino acids (i.e.,two Arg residues, one Lys, and one His). Therefore, the above peptidehas a net positive charge of three.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3 p_(m)is the largest number that is less than or equal to r+1. In thisembodiment, the relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) is asfollows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2 p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, suitably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a). Naturally occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides should also have a relationship betweenthe minimum number of aromatic groups (a) and the total number of netpositive charges at physiological pH (p_(t)) wherein 3a is the largestnumber that is less than or equal to p_(t)+1, except that when p_(t) is1, a may also be 1. In this embodiment, the relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (p_(t)) is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (2a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are suitably amidated with, for example, ammonia to form theC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N, N-dimethylamido,N, N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group. The free carboxylate groups of theasparagine, glutamine, aspartic acid, and glutamic acid residues notoccurring at the C-terminus of the aromatic-cationic peptides may alsobe amidated wherever they occur within the peptide. The amidation atthese internal positions may be with ammonia or any of the primary orsecondary amines described above.

In one embodiment, the aromatic-cationic peptide is a tripeptide havingtwo net positive charges and at least one aromatic amino acid. In aparticular embodiment, the aromatic-cationic peptide is a tripeptidehaving two net positive charges and two aromatic amino acids.

Aromatic-cationic peptides include, but are not limited to, thefollowing peptide examples:

TABLE 5 EXEMPLARY PEPTIDES 2′,6′-Dmp-D-Arg-2′,6′-Dmt-Lys-NH₂2′,6′-Dmp-D-Arg-Phe-Lys-NH₂ 2′,6′-Dmt-D-Arg-PheOrn-NH₂2′,6′-Dmt-D-Arg-Phe-Ahp(2-aminoheptanoicacid)-NH₂2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ 2′,6′-Dmt-D-Cit-PheLys-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheArg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D- Phe-Tyr-D-Arg-GlyAsp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-PheAsp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-Arg-2′,6′-Dmt-Lys-Phe-NH₂D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂ D-His-Glu-Lys-Tyr-D-Phe-ArgD-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-Asp-D-His-D-Lys- Arg-Trp-NH₂D-Tyr-Trp-Lys-NH₂ Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH₂ Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp. Gly-D-Phe-Lys-His-D-Arg-Tyr-NH₂His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂ Lys-D-Arg-Tyr-NH₂Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂ Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂Met-Tyr-D-Arg-Phe-Arg-NH₂ Met-Tyr-D-Lys-Phe-Arg Phe-Arg-D-His-AspPhe-D-Arg-2′,6′-Dmt-Lys-NH₂ Phe-D-Arg-HisPhe-D-Arg-Lys-Trp-Tyr-D-Arg-His Phe-D-Arg-Phe-Lys-NH₂Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe- NH₂Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D- Tyr-ThrThr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr- His-LysTyr-D-Arg-Phe-Lys-Glu-NH₂ Tyr-D-Arg-Phe-Lys-NH₂Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe Tyr-His-D-Gly-MetVal-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂

In one embodiment, the peptides have mu-opioid receptor agonist activity(i.e., they activate the mu-opioid receptor). Peptides, which havemu-opioid receptor agonist activity, are typically those peptides thathave a tyrosine residue or a tyrosine derivative at the N-terminus(i.e., the first amino acid position). Suitable derivatives of tyrosineinclude 2′-methyltyrosine (Mmt); 2′, 6′-dimethyltyrosine (2′6′-Dmt); 3′,5′-dimethyltyrosine (3′5′Dmt); N, 2′, 6′-trimethyltyrosine (Tmt); and2′-hydroxy-6′-methyltryosine (Hmt).

In one embodiment, a peptide that has mu-opioid receptor agonistactivity has the formula Tyr-D-Arg-Phe-Lys-NH₂. Tyr-D-Arg-Phe-Lys-NH₂has a net positive charge of three, contributed by the amino acidstyrosine, arginine, and lysine and has two aromatic groups contributedby the amino acids phenylalanine and tyrosine. The tyrosine ofTyr-D-Arg-Phe-Lys-NH₂ can be a modified derivative of tyrosine such asin 2′, 6′-dimethyltyrosine to produce the compound having the formula2′, 6′-Dmt-D-Arg-Phe-Lys-NH₂. 2′, 6′-Dmt-D-Arg-Phe-Lys-NH₂ has amolecular weight of 640 and carries a net three positive charge atphysiological pH. 2′, 6′-Dmt-D-Arg-Phe-Lys-NH₂ readily penetrates theplasma membrane of several mammalian cell types in an energy-independentmanner (Zhao, et al., J. Pharmacol Exp Ther., 304:425-432, 2003).

Alternatively, in other instances, the aromatic-cationic peptide doesnot have mu-opioid receptor agonist activity. For example, duringlong-term treatment, such as in a chronic disease state or condition,the use of an aromatic-cationic peptide that activates the mu-opioidreceptor may be contraindicated. In these instances, the potentiallyadverse or addictive effects of the aromatic-cationic peptide maypreclude the use of an aromatic-cationic peptide that activates themu-opioid receptor in the treatment regimen of a human patient or othermammal. Potential adverse effects may include sedation, constipation andrespiratory depression. In such instances an aromatic-cationic peptidethat does not activate the mu-opioid receptor may be an appropriatetreatment. Peptides that do not have mu-opioid receptor agonist activitygenerally do not have a tyrosine residue or a derivative of tyrosine atthe N-terminus (i.e., amino acid position 1). The amino acid at theN-terminus can be any naturally occurring or non-naturally occurringamino acid other than tyrosine. In one embodiment, the amino acid at theN-terminus is phenylalanine or its derivative. Exemplary derivatives ofphenylalanine include 2′-methylphenylalanine (Mmp), 2′,6′-dimethylphenylalanine (2′, 6′-Dmp), N, 2′, 6′-trimethylphenylalanine(Tmp), and 2′-hydroxy-6′-methylphenylalanine (Hmp).

An example of an aromatic-cationic peptide that does not have mu-opioidreceptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH₂.Alternatively, the N-terminal phenylalanine can be a derivative ofphenylalanine such as 2′, 6′-dimethylphenylalanine (2′6′-Dmp).Tyr-D-Arg-Phe-Lys-NH₂ containing 2′, 6′-dimethylphenylalanine at aminoacid position 1 has the formula 2′, 6′-Dmp-D-Arg-Phe-Lys-NH₂. In oneembodiment, the amino acid sequence of 2′, 6′-Dmt-D-Arg-Phe-Lys-NH₂ isrearranged such that Dmt is not at the N-terminus. An example of such anaromatic-cationic peptide that does not have mu-opioid receptor agonistactivity has the formula D-Arg-2′6′-Dmt-Lys-Phe-NH₂.

Suitable substitution variants of the peptides listed herein includeconservative amino acid substitutions. Amino acids may be groupedaccording to their physicochemical characteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe (F) Tyr(Y) Trp (W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group are generally more likely to alter thecharacteristics of the original peptide.

Examples of peptides that activate mu-opioid receptors include, but arenot limited to, the aromatic-cationic peptides shown in Table 6.

TABLE 6 Peptide Analogs with Mu-Opioid Activity Amino Amino Amino AminoAcid Acid Acid Acid C-Terminal Position 1 Position 2 Position 3 Position4 Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ Tyr D-Arg PheDab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂ 2′6′Dmt D-ArgPhe Lys-NH(CH₂)₂—NH- NH₂ dns 2′6′Dmt D-Arg Phe Lys-NH(CH₂)₂—NH- NH₂ atn2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-Cit Phe Lys NH₂ 2′6′Dmt D-Cit PheAhp NH₂ 2′6′Dmt D-Arg Phe Orn NH₂ 2′6′Dmt D-Arg Phe Dab NH₂ 2′6′DmtD-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Ahp(2-aminoheptanoic NH₂ acid)Bio-2′6′Dmt D-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Lys NH₂ 3′5′Dmt D-ArgPhe Orn NH₂ 3′5′Dmt D-Arg Phe Dab NH₂ 3′5′Dmt D-Arg Phe Dap NH₂ TyrD-Arg Tyr Lys NH₂ Tyr D-Arg Tyr Orn NH₂ Tyr D-Arg Tyr Dab NH₂ Tyr D-ArgTyr Dap NH₂ 2′6′Dmt D-Arg Tyr Lys NH₂ 2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′DmtD-Arg Tyr Dab NH₂ 2′6′Dmt D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg 2′6′Dmt LysNH₂ 2′6′Dmt D-Arg 2′6′Dmt Orn NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′DmtD-Arg 2′6′Dmt Dap NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg3′5′Dmt Lys NH₂ 3′5′Dmt D-Arg 3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt DabNH₂ Tyr D-Lys Phe Dap NH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂Tyr D-Lys Phe Orn NH₂ 2′6′Dmt D-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe DapNH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-LysPhe Orn NH₂ 3′5′Dmt D-Lys Phe Dab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′DmtD-Lys Phe Arg NH₂ Tyr D-Lys Tyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-LysTyr Dab NH₂ Tyr D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′DmtD-Lys Tyr Orn NH₂ 2′6′Dmt D-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂2′6′Dmt D-Lys 2′6′Dmt Lys NH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′DmtD-Lys 2′6′Dmt Dab NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg PhednsDap NH₂ 2′6′Dmt D-Arg Phe atnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂3′5′Dmt D-Lys 3′5′Dmt Orn NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′DmtD-Lys 3′5′Dmt Dap NH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Orn Phe Arg NH₂ TyrD-Dab Phe Arg NH₂ Tyr D-Dap Phe Arg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂2′6′Dmt D-Lys Phe Arg NH₂ 2′6′Dmt D-Orn Phe Arg NH₂ 2′6′Dmt D-Dab PheArg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂ 3′5′Dmt D-Arg Phe Arg NH₂ 3′5′DmtD-Lys Phe Arg NH₂ 3′5′Dmt D-Orn Phe Arg NH₂ Tyr D-Lys Tyr Arg NH₂ TyrD-Orn Tyr Arg NH₂ Tyr D-Dab Tyr Arg NH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′DmtD-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys 2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt Arg NH₂ 3′5′Dmt D-Dap 3′5′Dmt ArgNH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Lys 3′5′Dmt Arg NH₂ 3′5′DmtD-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe Lys NH₂ Mmt D-Arg Phe Orn NH₂ MmtD-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂ Tmt D-Arg Phe Lys NH₂ Tmt D-ArgPhe Orn NH₂ Tmt D-Arg Phe Dab NH₂ Tmt D-Arg Phe Dap NH₂ Hmt D-Arg PheLys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-Arg Phe Dab NH₂ Hmt D-Arg Phe DapNH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys Phe Orn NH₂ Mmt D-Lys Phe Dab NH₂Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe Arg NH₂ Tmt D-Lys Phe Lys NH₂ TmtD-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂ Tmt D-Lys Phe Dap NH₂ Tmt D-LysPhe Arg NH₂ Hmt D-Lys Phe Lys NH₂ Hmt D-Lys Phe Orn NH₂ Hmt D-Lys PheDab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-Lys Phe Arg NH₂ Mmt D-Lys Phe ArgNH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab Phe Arg NH₂ Mmt D-Dap Phe Arg NH₂Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe Arg NH₂ Tmt D-Orn Phe Arg NH₂ TmtD-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂ Tmt D-Arg Phe Arg NH₂ Hmt D-LysPhe Arg NH₂ Hmt D-Orn Phe Arg NH₂ Hmt D-Dab Phe Arg NH₂ Hmt D-Dap PheArg NH₂ Hmt D-Arg Phe Arg NH₂ Dab = diaminobutyric Dap =diaminopropionic acid Dmt = dimethyltyrosine Mmt = 2′-methyltyrosine Tmt= N,2′,6′-trimethyltyrosine Hmt = 2′-hydroxy,6′-methyltyrosine dnsDap =β-dansyl-L-α,β-diaminopropionic acid atnDap =β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of peptides that do not activate mu-opioid receptors include,but are not limited to, the aromatic-cationic peptides shown in Table 7.

TABLE 7 Peptide Analogs Lacking Mu-Opioid Activity Amino Amino AminoAmino Acid Acid Acid Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-ArgLys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-ArgPhe Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTrp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg TrpDmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg PheLys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl alanine

The amino acids of the peptides shown in Tables 5-7 may be in either theL- or the D-configuration.

The peptides may be synthesized by any of the methods well known in theart. Suitable methods for chemically synthesizing the protein include,for example, those described by Stuart and Young in Solid Phase PeptideSynthesis, Second Edition, Pierce Chemical Company (1984), and inMethods Enzymol., 289, Academic Press, Inc., New York (1997).

Cardiolipin Remodeling

Cardiolipin (cardiolipin) is an important component of the innermitochondrial membrane, where it constitutes about 20% of the totallipid composition. In mammalian cells, cardiolipin is found almostexclusively in the inner mitochondrial membrane where it is essentialfor the optimal function of enzymes involved in mitochondrialmetabolism.

Cardiolipin is a species of diphosphatidylglycerol lipid comprising twophosphatidylglycerols connected with a glycerol backbone to form adimeric structure. It has four alkyl groups and potentially carries twonegative charges. As there are four distinct alkyl chains incardiolipin, the molecule has the potential for great complexity.However, in most animal tissues, cardiolipin contains 18-carbon fattyalkyl chains with 2 unsaturated bonds on each of them (18:2). It hasbeen proposed that the 18:2 configuration is an important structuralrequirement for the high affinity of cardiolipin to inner membraneproteins in mammalian mitochondria. However, studies with isolatedenzyme preparations indicate that its importance may vary depending onthe protein examined.

Each of the two phosphates in cardiolipin can capture one proton.Although it has a symmetric structure, ionization of one phosphatehappens at different levels of acidity than ionizing both, with pK1=3and pK2>7.5. Hence, under normal physiological conditions (a pH ofapproximately 7.0), the molecule may carry only one negative charge.Hydroxyl groups (—OH and —O—) on the phosphate form stableintramolecular hydrogen bonds, forming a bicyclic resonance structure.This structure traps one proton, which is conducive to oxidativephosphorylation.

During the oxidative phosphorylation process catalyzed by Complex IV,large quantities of protons are transferred from one side of themembrane to another side causing a large pH change. Without wishing tobe bound by theory, it has been suggested that cardiolipin functions asa proton trap within the mitochondrial membranes, strictly localizingthe proton pool and minimizing pH in the mitochondrial intermembranespace. This function is thought to be due to the unique structure ofcardiolipin, which, as described above, can trap a proton within thebicyclic structure while carrying a negative charge. Thus, cardiolipincan serve as an electron buffer pool to release or absorb protons tomaintain the pH near the mitochondrial membranes.

In addition, cardiolipin has been shown to play a role in apoptosis. Anearly event in the apoptosis cascade involves cardiolipin. As discussedin more detail below, a cardiolipin-specific oxygenase producescardiolipin-hydroperoxides which causes the lipid to undergo aconformational change. The oxidized cardiolipin then translocates fromthe inner mitochondrial membrane to the outer mitochondrial membranewhere it is thought to form a pore through which cytochrome c isreleased into the cytosol. Cytochrome c can bind to the IP3 receptorstimulating calcium release, which further promotes the release ofcytochrome c. When the cytoplasmic calcium concentration reaches a toxiclevel, the cell dies. In addition, extra-mitochondrial cytochrome cinteracts with apoptotic activating factors, causing the formation ofapoptosomal complexes and activation of the proteolytic caspase cascade.

Other roles proposed for cardiolipin are: 1) participation instabilization of the physical properties of the membrane (Schlame, etal., 2000; Koshkin and Greenberg, 2002; Ma, et al., 2004), for example,membrane fluidity and osmotic stability and 2) participation in proteinfunction via direct interaction with membrane proteins (Schlame, et al.,2000; Palsdottir and Hunte, 2004). Cardiolipin has been found in tightassociation with inner membrane protein complexes such as the cytochromebc1 complex (complex III). As well, it has been localized to the contactsites of dimeric cytochrome c oxidase, and cardiolipin binding siteshave also been found in the ADP/ATP carrier (AAC; for review seePalsdottir and Hunte, 2004). Recent work also suggests a role ofcardiolipin in formation of respiratory chain supercomplexes(respirasomes).

The major tetra-acyl molecular species are 18:2 in each of the fourfatty acyl positions of the cardiolipin molecule (referred to as the18:2-18:2-18:2-18:2 cardiolipin species). Remodeling of cardiolipin isessential to obtain this enrichment of cardiolipin with linoleatebecause cardiolipin synthase has no molecular species substratespecificity for cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol. Inaddition, the species pattern of cardiolipin precursors is similarenough to imply that the enzymes of the cardiolipin synthetic pathwayare not molecular species-selective. Alterations in the molecularcomposition of cardiolipin are associated with various disease states.

Sengers Syndrome

Sengers syndrome is a rare autosomal recessive condition characterizedby, but not limited to, cataracts, hypertrophic cardiomyopathy, skeletalmyopathy, exercise intolerance, and lactic acidosis. The prevalence ofSengers syndrome is unknown; approximately 40 cases have been reportedto date in disparate locations throughout the world. The clinical coursevaries from severe forms that cause death in infancy to more benignforms that allow survival into adulthood. Approximately half of thereported patients die in the first year of life due to cardiac failure.Patients with milder cases have survived to their fifth decade of life.

Sengers syndrome is caused by mutations in the AGK gene. The AGK geneencodes the mitochondrial acylglycerol kinase, which plays a role in theassembly of adenine nucleotide translocator (ANT), an essentialcomponent of oxidative phosphorylation in mitochondria. AGK is part ofthe translocase of the inner membrane 22 (TIM22) complex, which plays arole in the carrier import pathway. As a lipid kinase, AGK is involvedin the conversion of monoacylglycerol (MAG) and diacylglycerol (DAG) tolysophosphatidic acid (LPA) and phosphatidic acid (PA). Phosphatidicacid is a precursor for the synthesis of mitochondrial cardiolipin,which is essential for mitochondrial structure and function. Thepathological mechanisms resulting in Sengers syndrome are unclear;however, they have been attributed to the role of AGK in lipidmetabolism.

Clinical features of Sengers syndrome include, but are not limited to,cataracts, hypertrophic cardiomyopathy, reduced cardiac function asassessed by ejection fraction, skeletal myopathy, exercise intolerance,lactic acidosis, neutropenia, tachydyspnea, nystagmus, eosinophilia,cervical meningocele, isolated Complex I deficiency, strabismus,hypotonia, hyporeflexia, delayed motor development, reduced AGKexpression, reduced mitochondrial levels of glutamate carrier 1 (GC1),reduced mitochondrial levels of adenine nucleotide transporter type 1(ANT1), reduced mitochondrial levels of adenine nucleotide transportertype 3 (ANT3), reduced mitochondrial levels of phosphate carrier (PiC),and reduced mitochondrial levels of translocase of the inner membrane 22(TIM22) subunits (hTim22, Tim29, and hTim9). Sengers syndrome maypresent in two forms, a neonatal lethal form or a chronic form.Hypertrophic cardiomyopathy is diagnosed at birth in approximately halfof the patients of both forms. Approximately half of the affectedindividuals die within the first year of life due to cardiac failure.Nystagmus, strabismus, hypotonia, hyporeflexia, and delayed motordevelopment are occasional features. Marked lactic acidosis occurs evenwith limited muscular exertion. Individuals who survive the neonatalperiod and infancy manifest the chronic form with stable cardiomyopathyand myopathy and have a normal intellect. Physical mobility is impaireddue to muscular weakness in most Sengers syndrome patients.

In some embodiments, treatment with a therapeutically effective amountof an aromatic-cationic peptide, such as D-Arg-2′6′-Dmt-Lys-Phe-NH₂, ora pharmaceutically acceptable salt thereof, such as acetate,hydrochloride or trifluoroacetate salt, increases the expression of AGKin a tissue or an organ in mammalian subjects that have suffered or areat risk of suffering Sengers syndrome.

In some embodiments, increasing AGK expression level is measured as aattenuation or reduction in the extent to which AGK expression isdecreased in a subject. In some embodiments, the AGK reduction isdecreased about 0.25 fold to about 0.5 fold, about 0.5 fold to about0.75 fold, about 0.75 fold to about 1.0 fold, or about 1.0 fold to about1.5 fold.

In some embodiments, administration of a therapeutically effectiveamount of an aromatic-cationic peptide, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt, to asubject in need thereof results in the treatment or prevention of one ormore signs or symptoms of Sengers syndrome including, but not limitedto, cataracts, hypertrophic cardiomyopathy, reduced cardiac function asassessed by ejection fraction, skeletal myopathy, exercise intolerance,lactic acidosis, neutropenia, tachydyspnea, nystagmus, eosinophilia,cervical meningocele, isolated Complex I deficiency, strabismus,hypotonia, hyporeflexia, delayed motor development, reduced AGKexpression, reduced mitochondrial levels of glutamate carrier 1 (GC1),reduced mitochondrial levels of adenine nucleotide transporter type 1(ANT1), reduced mitochondrial levels of adenine nucleotide transportertype 3 (ANT3), reduced mitochondrial levels of phosphate carrier (PiC),and reduced mitochondrial levels of translocase of the inner membrane 22(TIM22) subunits (hTim22, Tim29, and hTim9).

Therapeutic Methods

The following discussion is presented by way of example only, and is notintended to be limiting.

It is to be understood that increasing the expression level of AGK in asubject in need thereof (e.g., RNA and/or protein level) will reduce therisk, severity, presentation/onset of any number of negative physicaleffects. One aspect of the present technology includes methods oftreating reduced AGK expression in a subject diagnosed as having,suspected as having, or at risk of having reduced AGK expression levels.One aspect of the present technology includes methods of treatingSengers syndrome in a subject diagnosed as having, suspected as having,or at risk of having Sengers syndrome. In therapeutic applications,compositions or medicaments are administered to a subject suspected of,or already suffering from such a disease, such as, e.g., decreased AGKexpression levels or Sengers syndrome, in an amount sufficient to cure,or at least partially arrest, the symptoms of the disease, including itscomplications and intermediate pathological phenotypes in development ofthe disease.

Subjects suffering from decreased AGK expression levels or Sengerssyndrome can be identified by any or a combination of diagnostic orprognostic assays known in the art. For example, typical symptoms ofSengers syndrome include symptoms such as, e.g., cataracts, hypertrophiccardiomyopathy, lactic acidosis, skeletal muscle myopathy, nystagmus,and strabismus. In some embodiments, the subject may exhibit reducedlevels of AGK expression compared to a normal subject, which ismeasureable using techniques known in the art. In some embodiments, thesubject may exhibit one or more mutations in the AGK gene associatedwith Sengers syndrome, which are detectable using techniques known inthe art.

Prophylactic Methods

In one aspect, the present technology provides a method for preventingor delaying the onset of Sengers syndrome or symptoms of Sengerssyndrome in a subject at risk of having reduced levels of AGK expressioncompared to a normal subject. In some embodiments, the subject mayexhibit one or more mutations in the AGK gene associated with Sengerssyndrome, which are detectable using techniques known in the art.Subjects at risk for reduced AGK expression levels or Sengers syndromecan be identified by, e.g., any or a combination of diagnostic orprognostic assays known in the art. In prophylactic applications,pharmaceutical compositions or medicaments of aromatic-cationicpeptides, such as D-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceuticallyacceptable salt thereof, such as acetate, hydrochloride ortrifluoroacetate salt, are administered to a subject susceptible to, orotherwise at risk of Sengers syndrome, in an amount sufficient toeliminate or reduce the risk, lessen the severity, or delay the outsetof the disease, including biochemical, histologic and/or behavioralsymptoms of the disease, its complications and intermediate pathologicalphenotypes presenting during development of the disease. Administrationof a prophylactic aromatic-cationic can occur prior to the manifestationof symptoms characteristic of the disease or disorder, such thatsymptoms of the disease or disorder are prevented or, alternatively,delayed in their progression.

Subjects or at risk for reduced AGK expression levels or Sengerssyndrome may exhibit one or more of the following non-limiting riskfactors: cataracts, hypertrophic cardiomyopathy, reduced cardiacfunction as assessed by ejection fraction, skeletal myopathy, exerciseintolerance, lactic acidosis, neutropenia, tachydyspnea, nystagmus,eosinophilia, cervical meningocele, isolated Complex I deficiency,strabismus, hypotonia, hyporeflexia, delayed motor development, reducedAGK expression, reduced mitochondrial levels of glutamate carrier 1(GC1), reduced mitochondrial levels of adenine nucleotide transportertype 1 (ANT1), reduced mitochondrial levels of adenine nucleotidetransporter type 3 (ANT3), reduced mitochondrial levels of phosphatecarrier (PiC), and reduced mitochondrial levels of translocase of theinner membrane 22 (TIM22) subunits (hTim22, Tim29, and hTim9).

Determination of the Biological Effect of the Aromatic-CationicPeptide-Based Therapeutic

In various embodiments, suitable in vitro or in vivo assays areperformed to determine the effect of a specific aromatic-cationicpeptide-based therapeutic and whether its administration is indicatedfor treatment. In various embodiments, in vitro assays can be performedwith representative animal models, to determine if a givenaromatic-cationic peptide-based therapeutic exerts the desired effectincreasing AGK expression and/or preventing or treating Sengers syndromeand/or its signs and symptoms. Compounds for use in therapy can betested in suitable animal model systems including, but not limited torats, mice, chicken, cows, monkeys, rabbits, and the like, prior totesting in human subjects. Similarly, for in vivo testing, any of theanimal model system known in the art can be used prior to administrationto human subjects. In some embodiments, in vitro or in vivo testing isdirected to the biological function of D-Arg-2′6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate, hydrochlorideor trifluoroacetate salt.

Heart failure has been induced in different species with volumeoverload, pressure overload, fast pacing, myocardial ischemia,cardiotoxic drugs, or genetically modified models. Hypertension isassociated with an increased risk for the development of heart failure.In one mouse model, angiotensin II (Ang II) increases blood pressure andinduces cardiomyocyte hypertrophy, increased cardiac fibrosis, andimpaired cardiomyocyte relaxation. Infusion of angiotensin to mice bymini osmotic pump increases systolic and diastolic blood pressure,increases heart weight and left ventricular thickness (LVMI), andimpaired myocardial performance index (MPI). AGK expression levels aremonitored at various time points before, during and after heart failureinduction.

In a second illustrative mouse model, sustained high level expression ofGaq can lead to marked myocyte apoptosis, resulting in cardiachypertrophy and Heart failure by 16 weeks of age (D'Angelo, et al.,1998). The β-adrenergic receptors (βARs) are primarily coupled to theheterotrimeric G protein, Gs, to stimulate adenylyl cyclase activity.This association generates intracellular cAMP and protein kinase Aactivation, which regulate cardiac contractility and heart rate.Overexpression of Gaq leads to decreased responsiveness to β-adrenergicagonists and results in heart failure. AGK expression levels aremonitored at various time points before, during and after heart failureinduction.

Experimental constriction of the aorta by surgical ligation is alsowidely used as a model of heart failure. Transaortic constriction (TAC)results in pressure overload induced heart failure, with increase inleft ventricular (LV) mass. TAC is performed as described by Tamayski O,et al. (2004) using a 7-0 silk double-knot suture to constrict theascending aorta. After TAC, mice develop heart failure within a periodof 4 weeks. AGK expression levels are monitored at various time pointsbefore, during and after heart failure induction.

Modes of Administration and Effective Dosages

Any method known to those in the art for contacting a cell, organ ortissue with an aromatic-cationic peptide of the present technology, suchas D-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt, may beemployed. Suitable methods include in vitro, ex vivo, or in vivomethods. In vivo methods typically include the administration of anaromatic-cationic peptide, such as those described above, to a mammal,suitably a human. When used in vivo for therapy, the aromatic-cationicpeptides, such as D-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceuticallyacceptable salt thereof, such as acetate, hydrochloride ortrifluoroacetate salt, are administered to the subject in effectiveamounts (i.e., amounts that have desired therapeutic effect). The doseand dosage regimen will depend upon the degree of the infection in thesubject, the characteristics of the particular aromatic-cationic peptideused, e.g., its therapeutic index, the subject, and the subject'shistory.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of a peptide useful in the methods may be administeredto a mammal in need thereof by any of a number of well-known methods foradministering pharmaceutical compounds. The peptide may be administeredsystemically or locally.

The peptide may be formulated as a pharmaceutically acceptable salt. Theterm “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (e.g., salts having acceptable mammalian safety for agiven dosage regime). However, it is understood that the salts are notrequired to be pharmaceutically acceptable salts, such as salts ofintermediate compounds that are not intended for administration to apatient. Pharmaceutically acceptable salts can be derived frompharmaceutically acceptable inorganic or organic bases and frompharmaceutically acceptable inorganic or organic acids. In addition,when a peptide contains both a basic moiety, such as an amine, pyridineor imidazole, and an acidic moiety such as a carboxylic acid ortetrazole, zwitterions may be formed and are included within the term“salt” as used herein. Salts derived from pharmaceutically acceptableinorganic bases include ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic, manganous, potassium, sodium, and zincsalts, and the like. Salts derived from pharmaceutically acceptableorganic bases include salts of primary, secondary and tertiary amines,including substituted amines, cyclic amines, naturally-occurring aminesand the like, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically acceptable inorganicacids include salts of boric, carbonic, hydrohalic (hydrobromic,hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamicand sulfuric acids. Salts derived from pharmaceutically acceptableorganic acids include salts of aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (e.g., aspartic and glutamicacids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic,diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucuronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like. In some embodiments, the salt is an acetate, hydrochlorideor trifluoroacetate salt.

The aromatic-cationic peptides described herein, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt, can beincorporated into pharmaceutical compositions for administration, singlyor in combination, to a subject for the treatment or prevention of adisorder described herein. Such compositions typically include theactive agent and a pharmaceutically acceptable carrier. As used hereinthe term “pharmaceutically acceptable carrier” includes saline,solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. For convenience of thepatient or treating physician, the dosing formulation can be provided ina kit containing all necessary equipment (e.g., vials of drug, vials ofdiluent, syringes and needles) for a treatment course (e.g., 7 days oftreatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The aromatic-cationic peptide compositions can include a carrier, whichcan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thiomerasol, and the like. Glutathione and otherantioxidants can be included to prevent oxidation. In many cases, itwill be preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressurized container or dispenser,which contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer. Such methods include those described in U.S.Pat. No. 6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. In one embodiment, transdermal administration may be performedmy iontophoresis.

A therapeutic protein or peptide can be formulated in a carrier system.The carrier can be a colloidal system. The colloidal system can be aliposome, a phospholipid bilayer vehicle. In one embodiment, thetherapeutic peptide is encapsulated in a liposome while maintainingpeptide integrity. One skilled in the art would appreciate, there are avariety of methods to prepare liposomes. (See Lichtenberg, et al.,Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al., LiposomeTechnology, CRC Press (1993)). Liposomal formulations can delayclearance and increase cellular uptake (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). An active agent can also be loaded into aparticle prepared from pharmaceutically acceptable ingredientsincluding, but not limited to, soluble, insoluble, permeable,impermeable, biodegradable or gastroretentive polymers or liposomes.Such particles include, but are not limited to, nanoparticles,biodegradable nanoparticles, microparticles, biodegradablemicroparticles, nanospheres, biodegradable nanospheres, microspheres,biodegradable microspheres, capsules, emulsions, liposomes, micelles andviral vector systems.

The carrier can also be a polymer, e.g., a biodegradable, biocompatiblepolymer matrix. In one embodiment, the therapeutic peptide can beembedded in the polymer matrix, while maintaining protein integrity. Thepolymer may be natural, such as polypeptides, proteins orpolysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of, e.g., collagen, fibronectin, elastin,cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,and combinations thereof. In one embodiment, the polymer is poly-lacticacid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matricescan be prepared and isolated in a variety of forms and sizes, includingmicrospheres and nanospheres. Polymer formulations can lead to prolongedduration of therapeutic effect. (See Reddy, Ann. Pharmacother34(7-8):915-923 (2000)). A polymer formulation for human growth hormone(hGH) has been used in clinical trials. (See Kozarich and Rich, ChemicalBiology, 2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy, et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO96/40073 (Zale, et al.), and PCT publication WO 00/38651 (Shah, et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Such formulations can be preparedusing known techniques. The materials can also be obtained commercially,e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions (including liposomes targeted to specific cells withmonoclonal antibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

The therapeutic compounds can also be formulated to enhanceintracellular delivery. For example, liposomal delivery systems areknown in the art, see, e.g., Chonn and Cullis, “Recent Advances inLiposome Drug Delivery Systems,” Current Opinion in Biotechnology6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: SelectingManufacture and Development Processes,” Immunomethods, 4(3):201-9(1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery:Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995).Mizguchi, et al., Cancer Lett., 100:63-69 (1996), describes the use offusogenic liposomes to deliver a protein to cells both in vivo and invitro.

Dosage, toxicity and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds that exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to determine useful doses in humans accurately.Levels in plasma may be measured, for example, by high performanceliquid chromatography.

Typically, an effective amount of the aromatic-cationic peptides,sufficient for achieving a therapeutic or prophylactic effect, rangefrom about 0.000001 mg per kilogram body weight per day to about 10,000mg per kilogram body weight per day. Suitably, the dosage ranges arefrom about 0.0001 mg per kilogram body weight per day to about 100 mgper kilogram body weight per day. For example dosages can be 1 mg/kgbody weight or 10 mg/kg body weight every day, every two days or everythree days or within the range of 1-10 mg/kg every week, every two weeksor every three weeks. In one embodiment, a single dosage of peptideranges from 0.001-10,000 micrograms per kg body weight. In oneembodiment, aromatic-cationic peptide concentrations in a carrier rangefrom 0.2 to 2000 micrograms per delivered milliliter. An exemplarytreatment regime entails administration once per day or once a week. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the subject shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

In some embodiments, a therapeutically effective amount of anaromatic-cationic peptide may be defined as a concentration of peptideat the target tissue of 10¹² to 10′ molar, e.g., approximately 10′molar. This concentration may be delivered by systemic doses of 0.001 to100 mg/kg or equivalent dose by body surface area. The schedule of doseswould be optimized to maintain the therapeutic concentration at thetarget tissue, most preferably by single daily or weekly administration,but also including continuous administration (e.g., parenteral infusionor transdermal application).

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The subject treated in accordance with present methods can be any mammalor animal, including, for example, farm animals, such as sheep, pigs,cows, and horses; pet animals, such as dogs and cats; laboratoryanimals, such as rats, mice and rabbits. In a preferred embodiment, themammal is a human.

Combination Therapy with an Aromatic-Cationic Peptide and OtherTherapeutic Agents

There are no known treatments for Sengers syndrome. In most cases,surgery for cataracts and medical treatment for cardiac failure will berequired. Treatment is otherwise supportive and palliative. However, insome embodiments, the aromatic-cationic peptides, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt, may becombined with one or more additional agents for the prevention ortreatment of Sengers syndrome, such as agents for the prevention andtreatment of heart failure. Drug treatment for heart failure typicallyinvolves the administration of agents including, but not limited to, forexample, diuretics, angiotensin-converting enzyme (ACE) inhibitors,angiotensin II receptor blockers or inhibitors, angiotensin-receptorneprilysin inhibitors (ARNIs), I_(f) channel blockers or inhibitors,beta blockers, aldosterone antagonists, hydralazine and isosorbidedinitrate, diuretics, and digoxin (digitalis).

In one embodiment, an additional therapeutic agent is administered to asubject in combination with an aromatic cationic peptide, such that asynergistic therapeutic effect is produced. Therefore, lower doses ofone or both of the therapeutic agents may be used in treating Sengerssyndrome, resulting in increased therapeutic efficacy and decreasedside-effects.

In any case, the multiple therapeutic agents may be administered in anyorder or even simultaneously. If simultaneously, the multipletherapeutic agents may be provided in a single, unified form, or inmultiple forms (by way of example only, either as a single pill or astwo separate pills). One of the therapeutic agents may be given inmultiple doses, or both may be given as multiple doses. If notsimultaneous, the timing between the multiple doses may vary from morethan zero weeks to less than four weeks. In addition, the combinationmethods, compositions and formulations are not to be limited to the useof only two agents.

EXAMPLES

The present technology is further illustrated by the following examples,which should not be construed as limiting in any way.

Example 1—Use of Aromatic-Cationic Peptides in the Treatment of SengersSyndrome

This example demonstrates the effect of the aromatic-cationic peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ on cardiac function in a patient afflictedwith Sengers syndrome. In particular, the effects ofD-Arg-2′6′-Dmt-Lys-Phe-NH₂ on cardiomyopathy and Clinical GlobalImpression (CGI) were evaluated.

Methods

A patient with a homozygous AGK founder mutation (pIle348AsnfsTer38) wasdiagnosed with Sengers syndrome. Early in life, the patient was noted tohave severe cardiomyopathy, bilateral cataracts, and hypotonia.

The patient was treated with D-Arg-2′6′-Dmt-Lys-Phe-NH₂ forapproximately six months through a compassionate care protocol.Intravenous D-Arg-2′6′-Dmt-Lys-Phe-NH₂ (0.25 mg/kg/day administered overa 2-hour infusion (0.125 mg/kg/hr for 2 hour infusion) was initiated atthree months of age. After receiving pharmacokinetic results, the doseof D-Arg-2′6′-Dmt-Lys-Phe-NH₂ was increased to 0.50 mg/kg/day. WeeklyClinical Global Impression (CGI) evaluations and bi-weeklyechocardiograms were performed during the course of the six-monthtreatment.

Results

Overall, the patient improved from markedly ill to borderline ill(FIG. 1) and improved from the prior week at more than half (12/19) ofthe evaluations (Table 8). During the treatment period, the patient'scardiac function improved, as the initial ejection fraction as assessedby echocardiogram was poor, but improved in the first few weeks oftreatment and remained relatively stable thereafter. The patientexhibited no discernable side effects attributable to treatment withD-Arg-2′6′-Dmt-Lys-Phe-NH₂.

TABLE 8 CGI Short Form Results Severity of Global Date IllnessImprovement Comments June 12 Markedly ill Not assessed 6.225 kg(initiation 0.25 mg/kg/day) June 19 Moderately ill Minimally 6.540 kg,Better muscle tone, more spontaneous improved movements June 29Moderately ill Minimally Better muscle tone, more spontaneous improvedmovements, slight regress in myocardial hypertrophy July 4 Moderatelyill Minimally Better muscle tone, more spontaneous improved movementsJuly 11 Mildly ill Moderately Stable cardiac situation, HCM withoutoutflow improved obstruction, not in heart failure, growing adequately,feeding ~70% via NG tube July 18 Mildly ill Moderately 7.095 kg, Stablesituation, still needs NG tube improved feeds, dose increased tomorrow(elamipretide) POSTPONED July 25 Mildly ill No change 7.335 kg, Bettersleep, dose was not increased (dose doubled) (since last week) last weekas scheduled, increased today instead August 1 Mildly- MinimallyImproved feeding by mouth, improved motor Moderately ill improvedfunction, stable/mild impact of ECHO August 8 Mildly ill No changeUnchanged feeding, increased weight by 150 grams August 15 Mildly ill Nochange 7.660 kg, ECHO unchanged, mild neutropenia, off Nexium this weekperhaps a little worse self-feeding this week, now passing between arms,trying to stand with support/pull to stand August 29 Mildly illMinimally ECHO stable, status quo since last week improved September 6Mildly ill Minimally Better intake orally, vaccinated last week,improved neutrophils mild lowering, Lactate 12 at last test October 4Borderline ill No change ECHO stable HCM (hypertrophic cardiomyopathy)without outflow obstruction, no mitral regurgitation October 10Borderline ill No change Drinks only 20 mL of 160 mL of milk, muscleunchanged October 18 Mildly ill Minimally Sweating, increased vomiting,hardly any worse appetite October 25 Borderline ill Minimally Lessvomiting after carnitine was decreased, improved better appetiteNovember 1 Borderline ill Much improved Growing acceptably, cardiacfunction okay, no deterioration, carnitine 0.4 mL QID, CoQ10 - 1 capBID, Isbetol 2 mg PO AM, 1.75 mg PO PM November 14 Borderline ill Muchimproved ECHO: Septum 10 mm LV dia 27 mm/sys 16 mm PW 11 mm FS 40% No LVoutflow tract obstruction, mild mitral regurgitation HR 125/min November22 Borderline ill Much improved Stable, no significant change from lastweek Scale for assessing Severity of Illness: 0 = Not assessed; 1 =Normal, not at all ill; 2 = Borderline ill; 3 = Mildly ill; 4 =Moderately ill; 5 = Markedly ill; 6 = Severely ill; 7 = Among the mostextremely ill patients. Scale for assessing Global Improvement: 0 = Notassessed; 1 = Very much improved; 2 = Much improved; 3 = Minimallyimproved; 4 = No change; 5 = Minimally worse; 6 = Much worse; 7 = Verymuch worse.

CONCLUSIONS

The cardiac function in patients afflicted with Sengers syndrometypically deteriorates rapidly and most patients die as a result ofheart failure. These results show that aromatic-cationic peptides of thepresent technology, such as D-Arg-2′6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate, hydrochlorideor trifluoroacetate salt, are useful in the prevention and treatment ofSengers syndrome. Accordingly, the peptides are useful in methodscomprising administering aromatic-cationic peptides to a subject in needthereof for the treatment of Sengers syndrome.

Example 2—Effects of Aromatic-Cationic Peptides on the Expression ofMitochondrial Proteins in Sengers Syndrome Patient Cells and/or Tissue

This example demonstrates the effect of the aromatic-cationic peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ on levels of AGK expression, glutamatecarrier 1 (GC1), adenine nucleotide transporter type 1 (ANT1), adeninenucleotide transporter type 3 (ANT3), the mitochondrial phosphatecarrier (PiC), and translocase of the inner membrane 22 (TIM22)subunits, hTim22, Tim29, and hTim9, in tissues and fibroblasts fromSengers syndrome patients.

Methods

Primary Sengers syndrome patient fibroblasts are cultured in Dulbecco'smodified Eagles' medium (DMEM; Thermo Fisher Scientific) containing5%-10% (v/v) fetal bovine serum (FBS, In vitro Technologies) ortetracycline-reduced FBS (Clontech) and 0.01% (v/v)penicillin-streptomycin (Thermo Fischer Scientific) according to methodsknown in the art. The fibroblasts are cultured in the presence orabsence of D-Arg-2′6′-Dmt-Lys-Phe-NH₂ at varying concentrations ofD-Arg-2′6′-Dmt-Lys-Phe-NH₂ (10 nM-1 μM) for 24 hours. Mitochondria areisolated and mitochondrial proteins AGK, ANT1, ANT3, PiC, and TIM22subunits, hTim22, Tim29, and hTim9 are assessed by quantitative Westernblotting analysis according to methods known in the art.

Results

It is predicted that Sengers syndrome patient fibroblasts cultured withtherapeutically effective amounts of aromatic-cationic peptide, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt, willdisplay increased levels of one or more of AGK, ANT1, ANT3, PiC, orTIM22 subunits (hTim22, Tim29, and hTim9), as compared to untreatedSengers syndrome patient fibroblasts.

These results will show that aromatic-cationic peptides, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt, areuseful in the treatment of Sengers syndrome associated with reduced AGKlevels. Accordingly, the peptides of the present technology are usefulin methods comprising administering aromatic-cationic peptides tosubjects in need of normalization of AGK expression levels, such as, forexample, subjects having Sengers syndrome.

Example 3—Effects of Aromatic-Cationic Peptides on the Expression ofMitochondrial Proteins in AGK Knockout Cells

This example demonstrates the effect of the aromatic-cationic peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ on levels of glutamate carrier 1 (GC1),adenine nucleotide transporter type 1 (ANT1), adenine nucleotidetransporter type 3 (ANT3), the mitochondrial phosphate carrier (PiC),and translocase of the inner membrane 22 (TIM22) subunits, hTim22,Tim29, and hTim9, in an AGK knockout (AGK^(KO)) cell line.

Methods

The AGK^(KO) cell line is established according to Kang et al.,Molecular Cell 67:457-470 (2017). The AGK^(K)O cells are cultured in thepresence or absence of D-Arg-2′6′-Dmt-Lys-Phe-NH₂ at varyingconcentrations of D-Arg-2′6′-Dmt-Lys-Phe-NH₂ (10 nM-1 μM) for 24 hours.Mitochondria are isolated and mitochondrial proteins ANT1, ANT3, PiC,and TIM22 subunits (hTim22, Tim29, and hTim9) are assessed byquantitative Western blotting analysis according to methods known in theart.

Results

It is predicted that AGK^(KO) cells cultured with therapeuticallyeffective amounts of aromatic-cationic peptide, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt, willdisplay increased levels of one or more of ANT1, ANT3, PiC, or TIM22subunits (hTim22, Tim29, and hTim9), as compared to untreated controlAGK^(KO) cells.

These results will show that aromatic-cationic peptides, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt, areuseful in the treatment of Sengers syndrome associated with reduced AGKlevels. Accordingly, the peptides of the present technology are usefulin methods comprising administering aromatic-cationic peptides tosubjects in need of normalization of AGK expression levels, such as, forexample, subjects having Sengers syndrome.

Example 4—Use of Aromatic-Cationic Peptides in the Treatment of SengersSyndrome

This example will demonstrate the use of aromatic-cationic peptides,such as D-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptablesalt thereof, such as acetate, hydrochloride or trifluoroacetate salt,in the treatment of Sengers syndrome.

Methods

Sengers syndrome patients will receive daily administrations of atherapeutically effective amount of aromatic-cationic peptide, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt.Peptides may be administered orally, topically, systemically,intravenously, subcutaneously, intraperitoneally, intraocularly, orintramuscularly according to methods known in the art. Subjects will beevaluated weekly for the presence and/or severity of signs and symptomsassociated with Sengers syndrome, including, but not limited to, e.g.,cataracts, hypertrophic cardiomyopathy, reduced cardiac function asassessed by ejection fraction, skeletal myopathy, exercise intolerance,lactic acidosis, neutropenia, tachydyspnea, nystagmus, eosinophilia,cervical meningocele, isolated Complex I deficiency, strabismus,hypotonia, hyporeflexia, delayed motor development, reduced AGKexpression, reduced mitochondrial maximal respiration, reducedmitochondrial levels of glutamate carrier 1 (GC1), reduced mitochondriallevels of adenine nucleotide transporter type 1 (ANT1), reducedmitochondrial levels of adenine nucleotide transporter type 3 (ANT3),reduced mitochondrial levels of phosphate carrier (PiC), and reducedmitochondrial levels of translocase of the inner membrane 22 (TIM22)subunits (hTim22, Tim29, and hTim9). Treatments will be maintained untilsuch a time as symptoms of Sengers syndrome are ameliorated oreliminated.

Results

It is predicted that Sengers syndrome subjects receiving therapeuticallyeffective amounts of aromatic-cationic peptide, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt willdisplay reduced severity or elimination of symptoms associated withSengers syndrome.

These results will show that aromatic-cationic peptides, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate, hydrochloride or trifluoroacetate salt areuseful in the treatment of Sengers syndrome. Accordingly, the peptidesare useful in methods comprising administering aromatic-cationicpeptides to a subject in need thereof for the treatment of Sengerssyndrome.

EQUIVALENTS

The present technology is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this technology can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. Functionally equivalent methods and apparatuseswithin the scope of the present technology, in addition to thoseenumerated herein, will be apparent to those skilled in the art from theforegoing descriptions. Such modifications and variations are intendedto fall within the scope of the appended claims. The present technologyis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which such claims are entitled. It isto be understood that this technology is not limited to particularmethods, reagents, compounds compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method for treating or preventing Sengerssyndrome in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof.
 2. The method of claim 1, wherein the subject displays reducedlevels of acylglycerol kinase (AGK) expression compared to a normalcontrol subject.
 3. The method of any one of claims 1-2, wherein thepeptide is administered daily for 6 weeks or more.
 4. The method of anyone of claims 1-3, wherein the peptide is administered daily for 12weeks or more.
 5. The method of any one of claims 1-4, wherein thesubject has been diagnosed as having Sengers syndrome.
 6. The method ofclaim 5, wherein the Sengers syndrome comprises one or more ofcataracts, hypertrophic cardiomyopathy, reduced cardiac function asassessed by ejection fraction, skeletal myopathy, exercise intolerance,lactic acidosis, neutropenia, tachydyspnea, nystagmus, eosinophilia,cervical meningocele, isolated Complex I deficiency, strabismus,hypotonia, hyporeflexia, delayed motor development, reduced AGKexpression, reduced mitochondrial levels of glutamate carrier 1 (GC1),reduced mitochondrial levels of adenine nucleotide transporter type 1(ANT1), reduced mitochondrial levels of adenine nucleotide transportertype 3 (ANT3), reduced mitochondrial levels of phosphate carrier (PiC),and reduced mitochondrial levels of translocase of the inner membrane 22(TIM22) subunits (hTim22, Tim29, and hTim9).
 7. The method of any one ofclaims 1-6, wherein the subject is human.
 8. The method of any one ofclaims 1-7, wherein the peptide is administered orally, topically,systemically, intravenously, subcutaneously, intraocularly,intraperitoneally, or intramuscularly.
 9. The method of any one ofclaims 1-8, further comprising separately, sequentially orsimultaneously administering a cardiovascular agent to the subject. 10.The method of claim 9, wherein the cardiovascular agent is selected fromthe group consisting of: a diuretic, an angiotensin-converting enzyme(ACE) inhibitor, an angiotensin II receptor blocker or inhibitor, anangiotensin-receptor neprilysin inhibitor (ARNI), an I_(f) channelblocker or inhibitor, a beta blocker, an aldosterone antagonist, ahydralazine and isosorbide dinitrate, a diuretic, and digoxin.
 11. Themethod of any one of claims 1-10, wherein the pharmaceuticallyacceptable salt comprises acetate, hydrochloride or trifluoroacetatesalt.
 12. A method for increasing the expression of AGK in a mammaliansubject in need thereof, the method comprising: administering to thesubject a therapeutically effective amount of the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof.
 13. The method of claim 12, wherein the expression of AGK inthe subject is about 2-5 fold less than the level of AGK expression in anormal control subject.
 14. The method of any one of claims 12-13,wherein the peptide is administered daily for 6 weeks or more.
 15. Themethod of any one of claims 12-14, wherein the peptide is administereddaily for 12 weeks or more.
 16. The method of any one of claims 12-15,wherein the subject has been diagnosed as having, is suspected ofhaving, or is at risk of having Sengers syndrome.
 17. The method ofclaim 16, wherein the Sengers syndrome comprises one or more ofcataracts, hypertrophic cardiomyopathy, reduced cardiac function asassessed by ejection fraction, skeletal myopathy, exercise intolerance,lactic acidosis, neutropenia, tachydyspnea, nystagmus, eosinophilia,cervical meningocele, isolated Complex I deficiency, strabismus,hypotonia, hyporeflexia, delayed motor development, reduced AGKexpression, reduced mitochondrial levels of glutamate carrier 1 (GC1),reduced mitochondrial levels of adenine nucleotide transporter type 1(ANT1), reduced mitochondrial levels of adenine nucleotide transportertype 3 (ANT3), reduced mitochondrial levels of phosphate carrier (PiC),and reduced mitochondrial levels of translocase of the inner membrane 22(TIM22) subunits (hTim22, Tim29, and hTim9).
 18. The method of any oneof claims 12-17, wherein the subject is human.
 19. The method of any oneof claims 12-18, wherein the peptide is administered orally, topically,systemically, intravenously, subcutaneously, intraocularly,intraperitoneally, or intramuscularly
 20. The method of any one ofclaims 12-19, further comprising separately, sequentially orsimultaneously administering a cardiovascular agent to the subject. 21.The method of claim 20, wherein the cardiovascular agent is selectedfrom the group consisting of: a diuretic, an angiotensin-convertingenzyme (ACE) inhibitor, an angiotensin II receptor blocker or inhibitor,an angiotensin-receptor neprilysin inhibitor (ARNI), an I_(f) channelblocker or inhibitor, a beta blocker, an aldosterone antagonist, ahydralazine and isosorbide dinitrate, a diuretic, and digoxin.
 22. Themethod of any one of claims 12-21, wherein the pharmaceuticallyacceptable salt comprises acetate, hydrochloride or trifluoroacetatesalt.
 23. A method for reducing the risk of Sengers syndrome in amammalian subject having decreased expression of AGK compared to anormal control subject, the method comprising: administering to thesubject a therapeutically effective amount of the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof.
 24. The method of claim 23, wherein the pharmaceuticallyacceptable salt comprises acetate, hydrochloride or trifluoroacetatesalt.